The agonist activities of the putative antipsychotic agents,L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor

1. Dopamine D₄ receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced.
2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D_4.4 receptor (HEK293/D₄ cells). [³H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response.
3. [³H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D₄ cell homogenates (B_max 505±90 fmol mg⁻¹ protein, pK_D 9.5±0.1, n=3). Inhibition of specific [³H]-spiperone binding was observed with spiperone (pK_i 9.6±0.1, n=3), clozapine (pK_i 7.4±0.1, n=4), L-745,870 (pK_i 8.5±0.1, n=3) and U-101958 (pK_i 8.9±0.1, n=3). By contrast, raclopride was very weak (pK_i<5, n=3).
4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent fashion (E_max 71±2% inhibition of forskolin-stimulated levels, pEC₅₀ 8.7±0.1, n=10). This effect was mimicked by the dopamine D₂-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).
5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC₅₀) values of L-745,870 and U-101958 were 9.0±0.2 (n=4) and 8.7±0.3 (n=3), consistent with pK_i values determined in radioligand binding studies.
6. Dopamine, L-745,870 and U-101958 (up to 1 μM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells.
7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D₄ cells could be antagonized by spiperone (pK_B 8.2–8.8) and clozapine (pK_B 7.1), but not by raclopride (pK_B<5). None of these antagonists had any significant agonist activity at concentrations up to 10 μM.
8. These results show that the putative dopamine D₄ receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D_4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D₄ receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

     

Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2-dopamine-autoreceptors

1. Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D₂-dopamine receptors in the modulation of stimulation-induced (S-I) dopamine release from rat striatal slices incubated with [³H]-dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D₂ receptors since phorbol dibutyrate (PDB) and (−)-sulpiride, respectively, elevated S-I dopamine release.
2. The protein kinase C inhibitors polymyxin B (21 μM) and chelerythrine (3 μM) had no effect on stimulation-induced (S-I) dopamine release. However, when presynaptic dopamine D₂ receptors were blocked by sulpiride (1 μM), an inhibitory effect of both PKC inhibitors on S-I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D₂ receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down-regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished.
3. The inhibitory effect of the dopamine D₂/D₃ agonist quinpirole on S-I dopamine release was partially attenuated by PKC down-regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC-dependent as well as a PKC-independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down-regulation, suggesting that it operated exclusively through a PKC-independent mechanism.
4. These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally-released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.

     

κ1- and κ2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 μM)-induced release of [³H]-dopamine and [¹⁴C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional κ-opioid receptor subtypes in rat brain.
2. The κ receptor agonists (−)-ethylketocyclazocine ((−)-EKC), {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and the endogenous opioid peptide dynorphin A_1–13 caused a naloxone-reversible inhibition of NMDA-induced [³H]-dopamine release, with pD₂ values of about 9, 8.5 and 8.2, respectively, whereas both the μ agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and theδ agonist D-Pen²-D-Pen⁵-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A_1–13, {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and (−)-EKC on NMDA-induced [³H]-dopamine release were not changed by the δ₁/δ₂-opioid receptor antagonist naltrindole (up to a concentration of 1 μM), but reversed by the κ receptor antagonist nor-binaltorphimine (nor-BNI), with an IC₅₀ as low as 0.02 nM, indicating the involvement of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593-sensitive κ₁-opioid receptors.
3. NMDA-induced [¹⁴C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD₂ about 7.2), dynorphin A_1–13 (pD₂ 6.7) and EKC (pD₂ 6.2), but not by {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A_1–13 and (−)-EKC, on NMDA-induced [¹⁴C]-ACh release was antagonized by naltrindole with an IC₅₀ of 1 nM, indicating the involvement of δ-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A_1–13 and (−)-EKC on [¹⁴C]-ACh release were readily antagonized by nor-BNI with an IC₅₀ of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593-insensitive κ₂-opioid receptors in the inhibitory effects of dynorphin A_1–13 and (−)-EKC.
4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative κ₃-opioid receptor, antagonized the inhibitory effects of dynorphin A_1–13 and (−)-EKC on [³H]-dopamine and [¹⁴C]-ACh release as well as that of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 on [³H]-dopamine release, it displayed a low apparent affinity (IC₅₀ about 100 nM) in each case.
5. In conclusion, whereas activation of κ₁-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, κ₂ receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct κ-opioid receptor subtypes in the brain.

     

Direct dopamine D2-receptor-mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+-mobilizing agent

1. In CHO cells transfected with the rat dopamine D₂ receptor (long isoform), administration of dopamine per se elicited a concentration-dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC₅₀=25 nM). The partial D₂ receptor agonist, (−)-(3-hydroxyphenyl)-N-n-propylpiperidine [(−)-3-PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC₅₀=91 nM).
2. The AA-releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca²⁺ release, 8-(N N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), by excluding calcium from the medium, by the phospholipase A₂ (PLA₂) inhibitor, quinacrine, and by long-term pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, it was antagonized by the D₂ antagonists, raclopride and (−)-sulpiride–but not by (+)-sulpiride–and absent in sham-transfected CHO cells devoid of D₂ receptors.
3. The results obtained contrast to the previous notion that dopamine and other D₂ receptor agonists require the concomitant administration of calcium-mobilizing agents such as ATP, ionophore A-23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines.

     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

Effects of [3H]-BIDN,a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [³H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([³H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC₅₀, 35±3 nM) and dieldrin (IC₅₀, 93±7 nM), displaced [³H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [³H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [³H]-BIDN from rootworm membranes by a competitive mechanism.
3. Rat brain membranes were also shown to possess a population of saturable, specific [³H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [³H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA_B receptors were unaffected.
5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl⁻ channels to which a number of insecticidally-active molecules bind.

     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi

1. The release of neuronal [³H]acetylcholine (ACh) from isolated human bronchi after labelling with [³H]choline was measured to investigate the effects of prostanoids.
2. A first period of electrical field stimulation (S₁) caused a [³H]ACh release of 320±70 and 200±40 Becquerel (Bq) g⁻¹ in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S_n, n=2, 3, and 4) released less [³H]ACh, i.e. decreasing S_n/S₁ values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).
3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E₂, nocloprost, and sulprostone (EP₁ and EP₃ selective) inhibited evoked [³H]ACh release in a concentration dependent manner with IC₅₀ values between 4–14 nM and maximal inhibition of about 70%.
4. The inhibition of evoked [³H]ACh release by prostaglandin E₂, nocloprost and sulprostone was not affected by the DP-, EP₁- and EP₂-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA₂ values) at the respective receptors.
5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP₁-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [³H]ACh release.
6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [³H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [³H]ACh release.
7. In conclusion, applied prostanoids appear to inhibit [³H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP₃ subtype.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Characterization of [3H]-prostaglandin E2 binding to prostaglandin EP4 receptors expressed with Semliki Forest virus

1. The human prostaglandin EP₄ receptor has been expressed by use of the Semliki Forest virus system.
2. In cell membranes [³H]-prostaglandin E₂ ([³H]-PGE₂) bound to a high affinity site with a K_d of 1.12±0.3 nM and a B_max of 3.1±0.3 pmol mg⁻¹ protein.
3. In competition studies the rank order of potency for prostaglandins was PGE₂  = PGE₁  ≈#62;  PGF_2α =PGI₂.
4. The binding of [³H]-PGE₂ to cell membranes was inhibited by approximately 60% by the addition of guanylnucleotides, suggesting that this proportion of the receptors was G-protein coupled.
5. [³H]-PGE₂ binding was increased by greater than 200% by the addition of divalent cations, with little change in the IC₅₀ of PGE₂.
6. In saturation studies removal of divalent cations and addition of GTPγS resulted in a 65% reduction in the B_max with no change in the K_d. These results are consistent with the ligand labelling two states of the receptor R*, a high affinity state and R*G, a high affinity G protein coupled state.

     

LF 16.0335, a novel potent and selective nonpeptide antagonist of the human bradykinin B2 receptor

1. In the present paper, we describe the in vitro pharmacological properties of LF 16.0335 (1-[[3-[(2,4-dimethylquinolin-8-yl)oxymethyl]-2,4-dichloro-phenyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine), a novel and potent nonpeptide antagonist of the human bradykinin (BK) B₂ receptor.
2. LF 16.0335 displaced [³H]-BK binding to membrane preparations from CHO cells expressing the cloned human B₂ receptor, INT 407 cells and human umbilical vein with K_i values of 0.84±0.39 nM, 1.26±0.68 nM and 2.34±0.36 nM, respectively.
3. In saturation binding studies performed in INT 407 cell membranes in the presence or absence of LF 16.0335, B_max values of [³H]-BK were not significantly changed suggesting that LF 16.0335 behaves as a competitive antagonist.
4. LF 16.0335 had no affinity for the cloned human kinin B₁ receptor stably expressed in 293 cells. In addition, this compound at 1 μM did not significantly bind to a range of 40 different membrane receptors and eight ion channels except muscarinic M₂ and M₁ receptors for which an IC₅₀ value of 0.9 and 1 μM was obtained.
5. BK stimulates in a concentration-dependent manner phosphoinositosides (IPs) production in cultured INT 407 cells. Concentration-response-curves to BK were shifted to the right in the presence of LF 16.0335 (0.1 μM) without reduction of the maximum. LF 16.0335 inhibited the concentration-contraction curve to BK in the human umbilical vein giving a pA₂ value of 8.30±0.30 with a Schild plot slope that was not different from unity.
6. These results demonstrate that LF 16.0335 is a potent, selective and competitive antagonist of the human bradykinin B₂ receptor.

     

Comparative,general pharmacology of SDZ NKT 343, a novel,selective NK1 receptor antagonist

1. The in vitro and in vivo pharmacology of SDZ NKT 343 (2-nitrophenyl-carbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N-methylamide), a novel tachykinin NK₁ receptor antagonist was investigated.
2. SDZ NKT 343 inhibited [³H]-substance P binding to the human NK₁ receptor in transfected Cos-7 cell membranes (IC₅₀=0.62±0.11 nM). In comparison, in the same assay K_i values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13±0.04 nM, 0.96±0.20 nM, 0.15±0.06 nM and 1.77±0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK₁ receptors in whole forebrain membranes (IC₅₀=451±139 nM).
3. SDZ NKT 343 caused an increase in EC₅₀ as well as reduction in the number of binding sites (B_max) determined for [³H]-substance P, suggesting a non-competitive interaction at the human NK₁ receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca²⁺]_i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar⁹]SP sulphone-induced contraction of the guinea-pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible.
4. SDZ NKT 343 showed weak affinity to human NK₂ and NK₃ receptors in transfected Cos-7 cells (K_i of 0.52±0.04 μM and 3.4±1.2 μM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B₂ receptor the histamine H₁ receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage-activated Ca²⁺ and Na⁺currents in guinea-pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)-SDZ NKT 343 was about 1000 times less active at human NK₁ receptors expressed in Cos-7 cell membranes.
5. Contractions of the guinea-pig ileum by [Sar⁹]SP sulphone were inhibited by SDZ NKT 343 in a concentration-dependent manner, with an IC₅₀=1.60±0.94 nM, while the enantiomer (R,R)-SDZ NKT 343 was 100 times less active (IC₅₀=162±26 nM). In comparison, in the same assay IC₅₀ values for other NK₁ receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90±07 nM, 0.14±0.02 nM, 11.4±2.9 nM and 2.4±0.83 nM, respectively.
6. In anaesthetized guinea-pigs i.v. administered SDZ NKT 343 antagonized [Sar⁹]SP sulphone-evoked bronchoconstriction (70% reduction at 0.4 mg kg⁻¹, i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected.
7. In conclusion, SDZ NKT 343 is a highly selective NK₁ receptor antagonist with high potency at the human and guinea-pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK₁ receptor hyperfunction is involved.

     

Characterization of human recombinant somatostatin sst5 receptors mediating activation of phosphoinositide metabolism

1. We have functionally characterized the human recombinant somatostatin (SRIF) sst₅ receptor in Chinese hamster ovary-K1 (CHOsst₅) cells by measuring total [³H]-inositol phosphate ([³H]-InsPx) accumulation, in the presence of 10 mM LiCl, in cells labelled with [³H]-myo-inositol.
2. In CHOsst₅ cells, SRIF, SRIF-28 and the cyclic hexapeptide, L-362,855, produced time- and concentration-related increases in [³H]-InsPx accumulation, with similar potency (pEC₅₀ values of 6.5, 6.8 and 7.2, respectively). L-362,855 behaved as a partial agonist, producing approximately 30% of the SRIF maximum response. The other peptide analogues of SRIF, BIM-23027 and BIM-23056, were inactive as agonists.
3. Increasing concentrations of L-362,855 increased [³H]-InsPx accumulation and simultaneously produced rightward shifts of SRIF concentration-effect curves, with an estimated pK_p value of 7.6, confirming that it was acting as a partial agonist.
4. BIM-23056, but not BIM-23027, potently antagonized SRIF-induced [³H]-InsPx accumulation, with an estimated pK_B value of 7.4. BIM-23056 did not antagonize [³H]-InsPx accumulation induced by uridine 5′-triphosphate (UTP).
5. SRIF- but not UTP-induced [³H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01–100 ng ml⁻¹), indicating the involvement of pertussis toxin-sensitive G-proteins.
6. These findings show that the human recombinant sst₅ receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a specific antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.

     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

1. The present study describes the labelling of the nociceptin (NC) receptor, ORL₁, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [³H]-NC-NH₂, has a specific activity of 24.5 Ci mmol⁻¹
2. Saturation experiments revealed a single class of binding sites with a K_D value of 0.55 nM and B_max of 94 fmol mg⁻¹ of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: K_obs=0.104 min⁻¹; K₁=0.034 min⁻¹; T_1/2=20 min; K₊₁=0.07 min nM⁻¹.
3. Thermodynamic analyses indicated that [³H]-NC-NH₂ binding to the mouse ORL₁ is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ).
4. Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]NC(1–13)-NH₂ ([F/G]NC(1–13)-NH₂), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH=NC-NH₂=NC(1–13)-NH₂ >> NC(1–12)-NH₂ > NC(1–13)-OH >> NC(1–11)-NH₂, and for NC and NC(1–13)-NH₂ analogues: [Tyr¹]NC-NH₂ ⩾ [Leu¹]NC(1–13)-NH₂ ⩾ [Tyr¹]NC(1–13)-NH₂ ⩾ [F/G]NC(1–13)-NH₂ >> [Phe³]NC(1–13)-NH₂ > [DF/G]NC(1–13)-NH².
5. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [³H]-NC-NH₂ binding when applied at concentrations up to 10 μM indicating that this new radioligand interacts with a non opioid site, probably the ORL₁ receptor.

     

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist,in normal and diseased human tissues

1. We have synthesized a new low molecular weight, non-peptide radioligand, [¹²⁵I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ET_A receptor.
2. Analysis of saturation binding assays demonstrated that [¹²⁵I]-PD164333 bound with high affinity to a single population of receptors (n⩾3 individuals ±s.e.mean) in human aorta (K_D=0.26±0.08 nM; B_max=8.8±3.95 fmol mg^-1 protein), left ventricle from the heart (K_D=0.16±0.02 nM; B_max=34.2± 3.02 fmol mg^-1 protein) and kidney (K_D=1.24±0.16 nM; B_max=125.3±35.07 fmol mg^-1 protein). In each case Hill slopes were close to unity.
3. In kinetic experiments, the binding of [¹²⁵I]-PD164333 to ET_A receptors in sections of heart was time-dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K₁ of 2.66±0.213×10⁸ M^-1 min^-1, and a half-time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [¹²⁵I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min^-1, a half-time for dissociation of 216 min and a K_D calculated from these kinetic data of 0.01 nM.
4. Unlabelled PD164333 inhibited the binding of [¹²⁵I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a K_DET_A of 0.99±0.32 nM and K_DET_B of 2.41±0.22 μM, giving a selectivity of 2500 fold. ET_A-selective ligands competed monophasically for [¹²⁵I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, K_D=3.93 ±0.18 nM; {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 K_D=3.53±0.69 nM. In contrast, the ET_B selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding.
5. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA₂=8.84) and a slope of unity.
6. In the human brain, autoradiography revealed high levels of [¹²⁵I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [¹²⁵I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion.
7. These results show [¹²⁵I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ET_A receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.